Project Summary Alzheimer?s disease (AD) is a devastating neurodegenerative disease that particularly affects people over 65 years old. The major symptom of AD is a difficulty in forming long-term memories of newly acquired information, which requires proper development of synaptic connections between brain neurons at the cellular level. Arc is a neuronal protein specifically linked to the formation of long-term memory: Arc-knockout mice showed substantial deficits in long-lasting memories, but their short-term memories were not affected. However, a substantial knowledge gap exists to explain the molecular mechanism that allows Arc to regulate synaptic connections and affect memory formation, and this shortcoming in the literature has prevented Arc becoming a potential drug target for the treatment of AD. The long-term objective of the PI?s research program is to fill this gap and validate Arc as a new drug target specifically for the treatment of AD. The specific aims in this proposal address some of the fundamental problems in Arc research. These aims include: (1) the elucidation of the 3D structure of the full-length Arc protein; (2) the residue-specific characterization of Arc's binding sites for endophilin, dynamin, and presenilin; and (3) the determination of calcium ions' effect on the structure and function of Arc. These studies will help explain how Arc regulates AMPA receptor trafficking and ?-amyloid generation. They will also provide the knowledge base to understand Arc?s synaptic tagging, degradation, and nuclear transportation processes. To achieve Aim 1, the subdomain proteins of Arc will be produced using bacterial expression system, and their 3D structures will be determined using solution NMR techniques. The obtained structures will be used to help analyze the NMR spectra of the full-length Arc and construct its 3D structure. To achieve Aim 2, the NMR spectra of Arc will be compared with the spectra of Arc binding with endophilin/dynamin/presenilin. The spectra differences can be used to identify amino-acid residues that are responsible for Arc?s binding to different molecules. To achieve Aim 3, calcium ions will be titrated into Arc solutions with or without dynamin/endophilin/presenilin. The changes of the NMR spectra upon titration can be used to determine how calcium ions affect the structure and function of Arc. In our preliminary experiments, we successfully produced a C-terminal subdomain of the Arc protein and determined its 3D structure using NMR, which has laid the foundation for the proposed experiments. Achieving the proposed specific aims will provide new insights into the molecular mechanism of how Arc can modify synaptic connections to regulate memory formation. In the long term, this contributes to the field to use Arc as a drug target to treat AD.